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N~(#omega#)--硝基--L--精氨酸甲酯抑制吗啡镇痛耐受大鼠中脑和脊髓一氧化氮合酶/N--甲基--D--天冬氨酸受体表达上调

于玲, 薛富善, 李成文, 许亚超, 张国华, 刘鲲鹏, 刘毅, 孙海涛

中国医学科学院整形外科医院麻醉科.北京 100041

摘要

采用热甩尾测痛法观察全身应用非特异性一氧化氮合酶（nitric oxide synthase，NOS）抑制剂-----N~(#omega#)--硝基--L--精氨酸甲酯（L--NAME）对吗啡镇痛耐受形成的影响，并通过观察脊髓和中脑神经元型NOS（nNOS）和N--甲基--D--天冬氨酸（NMDA）受体亚单位表达的变化来阐释NO／NMDA受体在吗啡镇痛耐受形成中的作用。将36只健康成年Sprague--Dawley大鼠平均分为6组（每组6只）：1组为对照组，皮下注射生理盐水1ml；2、3、4、5和6组为处理组，分别皮下注射L--NAME10mg／kg、L--NAME20mg／kg、吗啡10mg／kg、L--NAME10mg／kg+吗啡10mg／kg、L--NAME20mg／kg+吗啡10mg／kg，每天2次。在注射前测量大鼠的热甩尾潜伏期（tail--flick latency，TFL）基础值，随后每天第一次给药50min后测量其TFL。d8最后一次给药80min后（除2组和5组之外）断头取脊髓和中脑，采用RT--PCR技术测量nNOS以及NMDA受体1A（NR1A）和2A（NR2A）亚单位的表达。结果显示，2组大鼠d1至d7的TFL与基础值相比无显著差异；3组d7时的TFL仍显著高于基础值；4组的TFL在d1时最高，d2至d6期间逐渐降低，d6时与基础值相比无显著差异：5组的TFL在实验过程中呈下降趋势，虽然d7时较d1有所降低，但是仍然显著高于基础值；6组的TFL变化趋势与5组相同。PT--PCR分析结果显示，与1组相比，3组脊髓和中脑的nNOS mRNA表达显著降低，但NR_(1A) mRNA和NR_(2A) mRNA表达无显著改变；4组的nNOS mRNA、NR_(1A) mRNA和NR_(2A) mRNA表达均显著高于1组。与4组相比，6组的nNOS mRNA、NR_(1A) mRNA和NR_(2A) mRNA表达均显著降低。结果提示，吗啡镇痛耐受大鼠脊髓和中脑的nNOS和NMDA受体表达增加，联合应用L--NAME可抑制长期应用吗啡所致的nNOS表达增加和NMDA受体上调，延缓吗啡镇痛耐受的形成。该研究结果提示，脊髓和中脑的NO／NMDA受体与吗啡镇痛耐受形成密切相关。

关键词： 吗啡; 镇痛耐受; 一氧化氮合酶; N-甲基-D-天冬氨酸受体

N~(#omega#)--nitro--L--arginine methyl ester inhibits the up--regulated expression of neuronal nitric oxide synthase/NMDA receptor in the morphine analgesia tolerance rats

Yu Ling, Xue Fushan, Li Chengwen, Xu Yachao, Zhang Guohua, Liu Kunpeng, Liu Yi, Sun Haitao

Department of Anesthesiology, Plastic Surgery Hospital, Chinese Academy of Medical Sciences.Beijing 100041

Abstract

The effect of systemic administration of nonspecific nitric oxide synthase inhibitor （N~(#omega#)-nitro-L-arginine methyl ester, L-NAME） on morphine analgesia tolerance was observed by using the thermal tail-flick method, and the roles of NO and NMDA receptors in morphine analgesia tolerance were evaluated on the basis of the expressions of nNOS mRNA, NR_(1A) mRNA and NR_(2A) mRNA in spinal cord and midbrain. Thirty-six healthy adult Sprague-Dawley rats were randomly divided into six groups （6 rats per group）. Group 1, control group, received a subcutaneous （s.c.） injection of normal saline （1 ml）; Groups 2, 3, 4, 5 and 6, the treatment groups received s.c. injection of L-NAME 10 mg/kg, L-NAME 20 mg/kg, morphine 10 mg/kg, L-NAME 10 mg/kg + morphine 10 mg/kg, and L-NAME 20 mg/kg + morphine 10 mg/kg, respectively. All rats received s.c. injections twice per day （8:00 and 17:00）. The tail-flick latency （TFL） was measured in each rat before the injection as a baseline value, and then TFL at 50 min after the 1st injection every day as the measuring values. The animals （except for groups 2 and 5） were decapitated at 80 min after the last injection on the 8th day. The spinal segments and midbrain were removed for analysis of nNOS mRNA, NR_(1A) mRNA and NR_(2A) mRNA expressions by the RT-PCR method. The results showed that TFL remained unchangeable in group 2 compared with baseline value during the 7-day observation, while increased significantly on the 7th day in group 3. In group 4, TFL was longest on the 1st day, then decreased gradually from the 2rid day to the 6th day, and restored to the baseline value on the 6th day. In group 5, TFL showed a decreasing tendency during the 7-day observation, but was still significantly longer than the baseline value on the 7th day. The changes of TFL obtained in group 6 were similar to those in group 5. The results of RT-PCR showed that as compared with group 1, nNOS mRNA expressions in spinal cord and midbrain were significantly down-regulated in group 3, but the expressions of the NR_(1A) mRNA and NR_(2A) mRNA in both groups were similar, while the nNOS mRNA, NR_(1A) mRNA and NR_(2A) mRNA expressions were all significantly up-regulated in group 4. As compared with group 4, the expressions of nNOS mRNA, NR_(1A) mRNA and NR_(2A) mRNA were significantly inhibited in group 6. These results suggest that the expressions of nNOS and NMDA receptors in spinal cord and midbrain were significantly up-regulated in the rats with morphine analgesia tolerance. Chronic co-administration of L-NAME could effectively inhibit the morphine-induced overexpressions of nNOS and NMDA receptors, and postpone the development of morphine analgesia tolerance. Based on the results of this study, it is concluded that NO/NMDA receptor in spinal cord and midbrain is closely related to the development of morphine analgesia tolerance.

Key words: Morphine;analgesia tolerance;nitric oxide synthase;NMDA receptor

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于玲, 薛富善, 李成文, 许亚超, 张国华, 刘鲲鹏, 刘毅, 孙海涛. N~(#omega#)--硝基--L--精氨酸甲酯抑制吗啡镇痛耐受大鼠中脑和脊髓一氧化氮合酶/N--甲基--D--天冬氨酸受体表达上调[J]. 生理学报 2006; 58 (6): .

Yu Ling, Xue Fushan, Li Chengwen, Xu Yachao, Zhang Guohua, Liu Kunpeng, Liu Yi, Sun Haitao. N~(#omega#)--nitro--L--arginine methyl ester inhibits the up--regulated expression of neuronal nitric oxide synthase/NMDA receptor in the morphine analgesia tolerance rats. Acta Physiol Sin 2006; 58 (6): (in Chinese with English abstract).